Figure 1 B-cell lymphoma 2, metallothionein-1, and Pim-2 overexpression decreases the embryoid body sizes of mouse embryonic stem cells. A and B: Mouse embryonic stem cells (ESCs) were trypsinized, aggregated into embryoid bodies (EBs) by hanging drop method, and then plated for terminal differentiation. Representative bright field images of mESC-T2, mESC-B-cell lymphoma 2, mESC-metallothionein-1, and mESC-PIM-2 (20 × magnification, scale bar = 100 μm) (A), and the average sizes of EBs are shown (B). Results are expressed as average sizes of EBs in each condition relative to the control. Data represent the average of three independent experiments and are reported as the mean ± standard error of the mean (^aP < 0.05). mESC: Mouse embryonic stem cell; BCL-2: B-cell lymphoma 2; MET-1: Metallothionein-1; EBs: Embryoid bodies.

Figure 2 B-cell lymphoma 2, metallothionein-1, and Pim-2 overexpression attenuates cardiac differentiation in mouse embryonic stem cells. The percentage of spontaneous and rhythmical beatings of differentiated cardiomyocytes was calculated in embryoid body-derived colonies on day 15 after plating for terminal differentiation. Results are expressed as the percentage of beating cardiomyocytes in each condition relative to the control. Data represent the average of three independent experiments and are reported as the mean ± standard error of the mean. ^aP < 0.05; ^bP < 0.01. mESC: Mouse embryonic stem cell; BCL-2: B-cell lymphoma 2; MET-1: Metallothionein-1.

Figure 3 B-cell lymphoma 2, metallothionein-1, and Pim-2 overexpression reduces the mRNA expression of troponin T, GATA4, and NKX2. 5 markers. Total RNA was collected after 14 d of differentiation and the mRNA expression of the three cardiac markers was analyzed using quantitative reverse transcription-polymerase chain reaction. The average of the normalized ratio of target genes/GAPDH was calculated. Data represent the average of three independent experiments and are reported as the mean ± standard error of the mean. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. mESC: Mouse embryonic stem cell; BCL-2: B-cell lymphoma 2; MET-1: Metallothionein-1.

Figure 4 B-cell lymphoma 2, metallothionein-1, and Pim-2 overexpression suppresses the protein expression of troponin T in mouse embryonic stem cells. Lysates of mouse embryonic stem cells were analyzed using immunofluorescence staining and western blot analysis. A: Representative immunofluorescence images stained with troponin T antibody (red) and the nuclear counterstain antifade Fluorogel II with 4’,6-diamidino-2-phenylindole (blue) (20 × magnification, scale bar = 100 μm). The images were acquired using confocal microscopy; B: Quantification of the red fluorescence intensity for troponin T. The quantification was performed using the Carl Zeiss ZEN 2012 image software; C: Immunoblots for troponin T. Bands were detected by enhanced chemiluminescence using the ChemiDoc MP Imaging System. GAPDH served as an internal control. mESC: Mouse embryonic stem cell; BCL-2: B-cell lymphoma 2; MET-1: Metallothionein-1; DAPI: 4’,6-diamidino-2-phenylindole.